This invention relates to the field of labels, particularly labels for diagnostic use. In particular, it relates to oligonucleotides that are modified to enhance the binding affinity of the oligonucleotides for complementary sequences and that in addition bear a readily detectable characteristic.
Sequence specific binding of oligonucleotides both to single stranded RNA and DNA and to duplex DNA is widely known. This phenomenon has been harnessed for a great variety of diagnostic, preparative and therapeutic purposes. Previously, one objective of research in this field has been to increase the affinity of such oligonucleotides for their complementary sequences. For example, Froehler et al. have described oligonucleotides containing 5-substituted pyrimidine bases that substantially increase the Tm for oligonucleotide binding to complementary bases (International Publication No. 92/10115).
Fluorescent cytosine derivatives are known for use in preparing labeled DNA probes. See Inoue et al., Jpn Kokai JP 62059293, (1987). In addition, fluorescent labeled nucleotides have been employed in DNA sequencing. See Prober et al., xe2x80x9cSciencexe2x80x9d 238:336-341 (1987).
1,3-Dihydro-2H-imidazo[4,5-b]-quinolin-2-one derivatives as phosphodiesterase inhibitors are disclosed by Raeymaekers et al. (EP 541,153).
An object of this invention is to increase the affinity of oligonucleotides for their complementary sequences.
Another object of this invention is to provide improved detectable labels for use in diagnostic assays.
A further object of this invention is to enhance diagnostic assays which employ oligonucleotides.
A still further object of this invention is to improve the therapeutic efficacy of oligonucleotides.
These and other objects of the invention will be apparent from consideration of the specification as a whole.
Structural formulas are designated as parenthetical numerals. It will be understood that designation of aromaticity with respect to carbocycles and heterocycles herein includes any highly resonant unsaturated ring structures Alternatively, placement of double bonds, where indicated, represents one potential structure for the depicted compound but will be understood to include other resonant states of the compound as well as protonated and charged species, only one of which may be shown.
In accordance with the objects, provided herein is a compound having the structure 
wherein
R1 is a binding partner, a linker or H;
a and b are 0 or 1, provided that the total of a and b is 0 or 1;
A is N or C;
X is S, O, xe2x80x94C(O)xe2x80x94, NH or NCH2R6;
Y is xe2x80x94C(O)xe2x80x94,
Z is taken together with A to form an aryl or heteroaryl ring structure comprising 5 or 6 ring atoms wherein the heteroaryl ring comprises a single O ring heteroatom, a single N ring heteroatom, a single S ring heteroatom, a single O and a single N ring heteroatom separated by a carbon atom, a single S and a single N ring heteroatom separated by a carbon atom, 2 N ring heteroatoms separated by a carbon atom, or 3 N ring heteroatoms at least two of which are separated by a carbon atom, and wherein the aryl or heteroaryl ring carbon atoms are unsubstituted with other than H or at least 1 nonbridging ring carbon atom is substituted with R6 or xe2x95x90O;
R3 is a protecting group or H;
R6 is independently H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, NO2, N(R3)2, Cxe2x89xa1N or halo, or an R6 is taken together with an adjacent R6 to complete a ring containing 5 or 6 ring atoms, and tautomers, solvates and salts thereof; and
provided that where a is 0, b is 1, and R1 is 
xe2x80x83in which
D2 is independently hydroxyl, blocked hydroxyl, mono, di or triphosphate, or an oligodeoxyribonucleotide otherwise containing only the bases A, G, T and C; and
D3is H or OH;
then Z is not unsubstituted phenyl.
When the binding partner R1 is an oligomer, embodiments of the compounds of this invention have structure (8) 
wherein
D is OH or blocked OH;
D1 is an oligonucleotide coupling group or OH
X1 is independently a phosphodiester linkage or a phosphodiester substitute linkage bonded to the 2xe2x80x2 or 3xe2x80x2 position of a furanose ring and the remaining 2xe2x80x2 or 3xe2x80x2 position is substituted with R21;
R21 is H, OH, F, xe2x80x94O-alkyl (C1-C12), xe2x80x94S-alkyl (C1-C12), OC3H5, or SC3H5;
n is an integer from 0 to 98; and
B is a purine or pyrimidine base or analogue thereof provided that at least one B has the structure 
wherein a, b, A, X, Y, Z, and the proviso are the same as for structure (1).
The compounds of structure (1) are made through several novel intermediates. The 4-pyridones are obtained from an intermediate having structure (2) 
wherein
R1 is H or a linker group;
J is an aryl or heteroaryl ring structure comprising 5 or 6 ring atoms wherein the heteroaryl ring comprises a single O ring heteroatom, a single N ring heteroatom, a single S ring heteroatom, a single O and a single N ring heteroatom separated by a carbon atom, a single S and a single N ring heteroatom separated by a carbon atom, or 2 N ring heteroatoms separated by a carbon atom, and wherein the aryl or heteroaryl ring carbon atoms are unsubstituted with other than H or at least 1 nonbridging ring carbon atom is substituted with R6; and
R6 is defined above;
and tautomers, salts and solvates thereof.
The 2-pyridones are synthesized from the intermediates of structures (3) and (6): 
wherein
R1 is H or a linker group;
R22 is C1-C3 alkyl; and
Jxe2x80x2 is an aryl or heteroaryl ring structure comprising 5 or 6 ring atoms wherein the heteroaryl ring comprises a single O ring heteroatom, a single N ring heteroatom, a single S ring heteroatom, a single O and a single N ring heteroatom separated by a carbon atom, a single S and a single N ring heteroatom separated by a carbon atom, or 2 N ring heteroatoms separated by a carbon atom, and wherein the aryl or heteroaryl ring carbon atoms are unsubstituted with other than H or at least 1 nonbridging ring carbon atom is substituted with C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, NO2, N(R3)2, or halo;
R3 is a protecting group or H;
and tautomers, solvates and salts thereof. 
wherein
A is independently S, O, N or CR6;
R6 is defined above; and
R26 is C1-C4 alkyl; and tautomers, salts and solvates thereof.
Phenoxazines and oxadiazines also are made from novel intermediate (5), as are pyridinopyrrolines, thiazines and oxazines. 
wherein
R1 is H or a linker group;
R24 is independently halo or C1-C2 haloalkyl;
R25 is independently xe2x80x94SH, xe2x80x94OH, xe2x95x90S or xe2x95x90O;
A is independently N or C; and
M, taken together with the radical xe2x80x94Axe2x80x94C(xe2x80x94R25), completes an aryl or heteroaryl ring structure comprising 5 or 6 ring atoms wherein the heteroaryl ring comprises a single O ring heteroatom, a single N ring heteroatom, a single S ring heteroatom, a single O and a single N ring heteroatom separated by a carbon atom, a single S and a single N ring heteroatom separated by a carbon atom, 2 N ring heteroatoms separated by a carbon atom, or 3 N ring heteroatoms at least two of which are separated by a carbon atom, and wherein the aryl or heteroaryl ring carbon atoms are unsubstituted with other than H or at least 1 nonbridging ring carbon atom is substituted with R6; and
R6 is defined above,
and tautomers, solvates and salts thereof.
The phenopyrrolines are made by the use of the intermediate of structure (4) 
wherein
R1 is H or a linker group;
J is an aryl or heteroaryl ring structure comprising 5 or 6 ring atoms wherein the heteroaryl ring comprises a single O ring heteroatom, a single N ring heteroatom, a single S ring heteroatom, a single O and a single N ring heteroatom separated by a carbon atom, a single S and a single N ring heteroatom separated by a carbon atom, or 2 N ring heteroatoms separated by a carbon atom, and wherein the aryl or heteroaryl ring carbon atoms are unsubstituted with other than H or at least 1 nonbridging ring carbon atom is substituted with R6; and
R6 is defined above;
R23 is a protecting group;
and tautomers, salts and solvates thereof.